Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial "burst" phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pKa values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.